Saintopin derivatives

ABSTRACT

The present invention relates to Saintopin derivatives represented by the following formula (I): ##STR1## wherein R 1  is hydrogen and R 2  is SO 2  OH (UCE 1022), or R 1  is acetyl and R 2  is H (Saintopin E), which are useful as antibacterial and anti-tumor agents.

This application is a 371 of PCT/JP93/01680.

TECHNICAL FIELD

The present invention relates to Saintopin derivatives which haveantibacterial and anti-tumor activity and are useful as antibacterialand anti-tumor agents.

BACKGROUND ART

Heretofore, some compounds such as anthracycline have been reported asantibiotics having anthraquinone skeleton CRC Handbook of AntibioticCompounds, 3, 61 (1981)!.

Further, UCT 1003 (Saintopin) having anti-tumor activity which isrepresented by the following formula A: ##STR2## has also been knownBiochemistry, 30, 5838-5845 (1991); Japanese Published Unexamined PatentApplication No. 200655/90!.

DISCLOSURE OF THE INVENTION

The present invention relates to Saintopin derivatives havingantibacterial and anti-tumor activity which are represented by thefollowing formula (I): ##STR3## wherein R¹ is hydrogen and R² is SO₂ OH(hereinafter, the compound given by the definition is referred to as UCE1022), or R¹ is acetyl and R² is hydrogen (hereinafter, the compoundgiven by the definition is referred to as Saintopin E).

The compounds described above can be produced by culturing amicroorganism belonging to the genus Paecilomyces.

The present invention is described in detail below.

The compound UCE 1022 has the following physicochemical properties.

(1) Molecular weight: 418

(2) Molecular formula: C₁₈ H₁₀ O₁₀ S

(3) Mass spectrum:

Secondary ion mass spectrum, negative mode (matrix: m-nitrobenzylalcohol): m/z; 417(M--H)⁻

High resolution FAB mass spectrum, negative mode (matrix: m-nitrobenzylalcohol): m/z

Found: 416.9919

Calculated for C₁₈ H₉ O₁₀ S: 416.9917

(4) UV absorption spectrum (measured in methanol) λmax nm (ε); 241(22,000), 274 (27,500), 306 (16,100), 339 (9,800), 483 (12,300)

(5) IR absorption spectrum (measured by the KBr method): νmax cm⁻¹ ;3360, 3210, 1625, 1600, 1400, 1330, 1265, 1235, 1045

(6) ¹³ C-NMR spectrum (125 MHz, methanol-d₄ solution): δ ppm; 108.3,109.2, 109.3, 111.4, 111.6, 114.8, 116.3, 121.9, 130.0, 137.5, 141.2,154.3, 161.9, 166.4, 166.5, 166.6, 183.4, 190.8

(7) ¹ H-NMR spectrum (500 MHz, methanol-d₄ solution): δ ppm; 6.57(1H,d), 7.09(1H, d), 7.21(1H, d), 7.41(1H, d), 7.96 (1H, s)

(8) Solubility:

Soluble in water, methanol and ethanol, and sparingly soluble inn-hexane (UCT 1003 is sparingly soluble in water)

(9) Color reaction:

Positive to the iodine and cerium sulfate-sulfuric acid tests

(10) Color and form of the substance:

Reddish purple powder

(11) Thin layer chromatography:

Rf value is 0.25 in silica gel thin layer chromatography (HPTLC plateArt. 5715, produced by Merck & Co., Inc.) with a developing solventcomposed of n-hexane:ethyl acetate:methanol:acetic acid (6:4:1:1, v/v)(The spot of UCE 1022 is detectable by the absorption in the visible andUV regions).

Saintopin E has the following physicochemical properties.

(1) Molecular weight: 380

(2) Molecular formula: C₂₀ H₁₂ O₈

(3) Mass spectrum:

High resolution FAB mass spectrum (matrix: m-nitrobenzyl alcohol): m/z

Found: 381.3223 (M+H)⁺

Calculated for C₂₀ H₁₃ O₈ : 381.3214

(4) UV absorption spectrum (measured in methanol) λmax nm (ε); 290(17,100), 338 (8,100), 566 (7,300)

(5) IR absorption spectrum (measured by the KBr method): νmax cm⁻¹ ;3431, 3230, 1622, 1564, 1437, 1387, 1269, 1163

(6) ¹ H-NMR spectrum (500 MHz, DMSO-d₆ solution): δ ppm; 14.10(1H,br.s), 13.43(1H, br.s), 10.76(1H, br.s), 7.46(1H, s), 7.04(1H, d, 2.1Hz), 6.53(1H, d, 2.1 Hz), 6.34(1H, s), 2.66(3H, s)

(7) Solubility:

Readily soluble in dimethyl sulfoxide (DMSO), methanol and acetone,soluble in chloroform and ethyl acetate, and sparingly soluble in waterand n-hexane

(8) Color and form of the substance:

Bluish purple powder

(9) Thin layer chromatography:

Rf value is 0.50 in silica gel thin layer chromatography (HPTLC plateArt. 5715, produced by Merck & Co., Inc.) with a developing solventcomposed of n-hexane:ethyl acetate:methanol:acetic acid (6:4:1:1, v/v)

(10) High performance liquid chromatography:

Retention time; 8.56 min. (Column; YMC AM312 ODC, produced by YMCCorporation: Flow rate; 1 ml/min.:

Mobile phase; a 70% methanol solution containing 5 mM ammonium acetate)

The biological activities of Compounds (I) are described below.

TEST EXAMPLE 1 Antibacterial activity

The minimum inhibitory concentration (MIC) against the growth of variousbacteria is shown in Table 1. The antibacterial activity was determinedby the agar dilution method using a medium (pH 7) which comprises 3 g/lBacto-Tryptone (produced by Difco Laboratories), 3 g/l meat extract, 1g/l yeast extract, 1 g/l glucose and 16 g/l agar.

                  TABLE 1    ______________________________________    Bacteria tested                   MIC (μg/ml) (UCE 1022)    ______________________________________    Staphylococcus aureus                   0.52    ATCC 6538P    Enterococcus faecium                   0.52    ATCC 10541    ______________________________________

TEST EXAMPLE 2 Anti-tumor activity against HeLaS₃ cells

HeLaS₃ cells (ATCC HTB22) were suspended in a medium comprising 10%fetal calf serum, 2 mM glutamine and MEM medium (produced by NipponPharmaceutical Co., Ltd.) (hereinafter referred to as medium A) to aconcentration of 3×10⁴ cells/ml. The cell suspension was put into wellsof a 96-well microtiter plate in an amount of 0.1 ml per well. The cellsin the plate were cultured at 37° C. for 20 hours in a CO₂ -incubator.Subsequently, the test compound appropriately diluted with medium A wasadded to the wells in an amount of 0.1 ml/well. The cells were furthercultured at 37° C. for 72 hours in the CO₂ -incubator, and then theculture supernatant was removed. To the residue was added a mediumcomprising medium A and 0.02% Neutral Red in an amount of 0.1 ml perwell, followed by culturing at 37° C. for one hour in the CO₂-incubator, whereby the cells were stained. After removal of the culturesupernatant, the residue was washed once with physiological saline. Thepigment was extracted with 0.001N hydrochloric acid/30% ethanol, and theabsorbance at 550 nm was measured by using a microplate reader. Theconcentration of the test compound at which the growth of the cells isinhibited by 50% (IC₅₀) was calculated by comparing the absorbance ofuntreated cells with those of the cells treated with the test compoundat known concentrations. The result is shown in Table 2.

                  TABLE 2    ______________________________________    Test compound   IC.sub.50 (μM)    ______________________________________    UCE 1022        6.1    ______________________________________

TEST EXAMPLE 3 Growth inhibition against BALB 3T3/H-ras cells

BALB 3T3/H-ras cells were suspended in F10 medium comprising 10% fetalcalf serum, 100 units/ml penicillin and 100 mg/ml streptomycin (aproduct by GIBCO Company; hereinafter referred to as medium B) to aconcentration of 2×10⁴ cells/ml. The cell suspension was put into wellsof a 96-well microtiter plate in an amount of 0.1 ml per well. The cellsin the plate were cultured at 37° C. for 20 hours in a CO₂ -incubator.Subsequently, the test compound appropriately diluted with medium B wasadded to the wells in an amount of 0.05 ml per well. The cells werefurther cultured at 37° C. for 72 hours in the CO₂ -incubator. Thesystem was treated in the same manner as in Test Example 2 to calculatethe concentration of the test compound at which the growth of the cellsis inhibited by 50% (IC₅₀). The result is shown in Table 3.

                  TABLE 3    ______________________________________    Test compound   IC.sub.50 (μM)    ______________________________________    Saintopin E     2.4    ______________________________________

The process for producing Compounds (I) is described below.

UCE 1022, which is included in Compounds (I), can be obtained byculturing a microorganism belonging to the genus Paecilomyces and havingthe ability to produce UCE 1022 in a medium, allowing UCE 1022 toaccumulate in the culture, and recovering UCE 1022 from the culture.

As the UCE 1022-producing strains of the present invention, any strainswhich belong to the genus Paecilomyces and have the ability to produceUCE 1022 can be used. In addition, any mutants of such strains which areobtained by various artificial mutation methods such as UV irradiation,X ray irradiation and treatment with mutagens or by spontaneous mutationmay also be used in the present invention, insofar as they have theability to produce UCE 1022. A typical example of a suitable strain isUOE 1022 strain which has been isolated from soil by the presentinventors.

The mycological properties of UOE 1022 strain are as follows.

1. Macroscopic observation

When UOE 1022 strain is cultured at 25° C. on a malt extract agarmedium, the diameter of a colony reaches about 28 mm on the seventh dayfrom the start of the culturing. The surface of the colony is floccoseand shows a white color. The center of the back side of the colony showsan orange color, and the edge thereof shows a pale yellow color. In theculture medium, the dissolution of a soluble pale yellow pigment isobserved.

When this strain is cultured at 25° C. on a potato glucose agar medium,the diameter of a colony reaches 26 to 27 mm on the seventh day from thestart of the culturing. The surface of the colony is floccose, and thecenter thereof shows a lilac color or a purple color and the edge awhite color to a gray color. The back side of the colony shows a lemoncolor. In the culture medium, the dissolution of a soluble pale yellowpigment is observed.

The optimum growth temperature for this strain is in the range of 11° to34° C., and most preferably about 23° C. The pH range that allows itsgrowth is 3 to 10.5, and the optimum growth pH is around 6.

2. Observation under optical microscope

UOE 1022 strain is cultured at 25° C. on a malt extract agar medium andis observed under the optical microscope. The results are as follows.

Hyphae are septate and smooth and branch well. Conidiophores arise fromthe hyphae, are colorless and smooth, and branch in the upper part in averticillate form or irregularly. On the top of each branch of theconidiophores, two to four phialides are formed. In some cases, one tofour phialides are formed directly on the top of each conidiophore. Thephialides are colorless, smooth and lageniform, and taper off to apoint. They are 7 to 12 μm long, and 2 to 3 μm wide at the part near itsjoint, tapering to about 0.5 μm wide. The conidial ontogeny isenteroblastic. Phialoconidia are unicellular and fusiform or limoniformin shape. They are colorless and smooth, and are 3 to 4.5 μm long and1.5 to 2.5 μm wide. The conidia develop in the form of a long chain fromthe top of the phialide. Only the anamorph as described above isobserved for this strain, with no teleomorph being observed at all.

A taxonomical study of this strain based on the above mycologicalproperties according to "The Genera of Fungi Sporulating in PureCulture, 2nd Ed., Cramer, Vaduz, J. A. von Arx, 1974" revealed that itbelongs to the genus Paecilomyces, which falls under hyphomycetes.

The strain was named Paecilomyces sp. UOE 1022 by the present inventorsand was deposited with the National Institute of Bioscience and HumanTechnology with accession number FERM BP-4066 on Nov. 5, 1992.

The process for producing Saintopin E is described below.

Saintopin E can be obtained by culturing a microorganism belonging tothe genus Paecilomyces and having the ability to produce Saintopin E ina medium, allowing Saintopin E to accumulate in the culture, andrecovering Saintopin E from the culture.

As the Saintopin E-producing strains of the present invention, anystrains which belong to the genus Paecilomyces and have the ability toproduce Saintopin E can be used. In addition, any mutants of suchstrains which are obtained by various artificial mutation methods suchas UV irradiation, X ray irradiation and treatment with mutagens or byspontaneous mutation may also be used in the present invention, insofaras they have the ability to produce Saintopin E. A typical example of asuitable strain is SPC-13780 strain.

The strain has been deposited with the Fermentation Research Institute,Agency of Industrial Science and Technology under the Budapest Treatywith accession number FERM BP-2256 (date of original deposit: Jan. 24,1989) (Japanese Published Unexamined Patent Application No. 00655/90).

The culturing methods for UCE 1022-producing strains and SaintopinE-producing strains are as follows.

For the culturing of the Saintopin derivatives-producing strains used inthe present invention, conventional methods for culturing molds aregenerally employed. As the medium, either a synthetic medium or anatural medium may be used insofar as it appropriately contains carbonsources, nitrogen sources and inorganic substances which can beassimilated by the strains employed and the growth- andproduction-promoting substances required.

As the carbon sources, glucose, starch, dextrin, mannose, fructose,sucrose, lactose, xylose, arabinose, mannitol, molasses, etc. can beused alone or in combination. In addition, hydrocarbons, alcohols,organic acids, etc. may also be used according to the assimilability ofthe microorganism employed. As the nitrogen sources, ammonium chloride,ammonium nitrate, ammonium sulfate, sodium nitrate, urea, peptone, meatextract, yeast extract, dry yeast, corn steep liquor, soybean powder,casamino acid, etc. can be used alone or in combination. If necessary,inorganic salts such as sodium chloride, potassium chloride, magnesiumsulfate, magnesium phosphate, calcium carbonate, potassiumdihydrogenphosphate, ferrous sulfate, calcium chloride, manganesesulfate, zinc sulfate, copper sulfate, etc. may be added. In addition,trace ingredients that promote the growth of the strain employed and theproduction of the Saintopin derivatives may also be added to the medium.

As the method of culturing, liquid culture, especially submergedstirring culture, is preferably employed. Culturing is carried out at16° to 37° C., preferably 25° to 32° C., and at pH 4 to 10, preferably 6to 8. In general, by culturing for 1 to 7 days, the desired substance isproduced and accumulated in the culture. In order to adjust the pH ofthe medium, aqueous ammonia, ammonium carbonate solution, etc. are used.When the amount of the product in the culture reaches the maximum, theculturing is discontinued.

For the isolation and purification of the desired substance from theculture, an ordinary method for isolating a microbial metabolite fromthe culture can be utilized. For example, the culture is separated intoculture filtrate and microbial cells by filtration. The microbial cellsare extracted with methanol, acetone, or the like. Then, the extract ismixed with the culture filtrate, and the resulting mixture is passedthrough a column of polystyrene adsorbent such as Diaion HP20 (producedby Mitsubishi Kasei Corporation) to adsorb the active component,followed by elution with ethyl acetate, acetone, or the like. The eluateis concentrated, and the concentrate is subjected to silica gel columnchromatography, high performance liquid chromatography, and the like toobtain the desired substance. During the culture and purification steps,the desired substance can be traced by the UV absorbance of thesubstance.

Examples of the present invention are shown below.

BEST MODE FOR CARRYING OUT THE INVENTION Example 1

Paecilomyces sp. UOE 1022 strain (FERM BP-4066) was used as the seedstrain. The strain was inoculated into 300 ml of a seed medium havingthe following composition in a 2-l Erlenmeyer flask, and cultured withshaking at 25° C. for 48 hours.

Composition of the seed medium (in 1 l): 5 g of peptone (KyokutoPharmaceutical Co. Ltd.), 5 g of dry yeast "Ebios", 10 g of glucose, 200ml of V8 vegetable juice (Campbell Japan), 0.5 g of Mg₃ (PO₄)₂ ·8H₂ O(pH 6.0 before sterilization)

The resulting seed culture was transferred into 18 l of a fermentationmedium having the following composition in a 30-l jar fermentor at therate of 5% (by volume), and culturing was carried out at 25° C. withstirring and aeration (rotation: 300 rpm, aeration: 18 l/min.).

Composition of the fermentation medium (in 1 l): 50 g of soluble starch,30 g of corn steep liquor (Japan Maize Products Co., Ltd.), 0.5 g of KH₂PO₄, 0.5 g of MgSO₄ ·7H₂ O, 0.5 g of Mg₃ (PO₄)₂ ·8H₂ O (pH 7.0 beforesterilization, adjusted with NaOH)

Culturing was carried out for 144 hours without controlling the pH ofthe medium.

After the completion of culturing, 7.5 l of the fermentation culture wasfiltered and 10 l of methanol was added to the cell fraction, followedby stirring, whereby UCE 1022 was extracted. To the methanol extract wasadded 30 l of water, and the mixture was passed through a column packedwith 0.5 l of a non-ionic porous resin, Diaion HP20 (produced byMitsubishi Kasei Corporation), to adsorb the active substance. Afterimpurities were eluted with methanol/water (30:70, v/v), the activesubstance was eluted with 1.5 l of methanol/water (50:50, v/v). Theactive fraction eluted was concentrated, and the concentrate was appliedto a column packed with 0.5 l of silica gel (BW 300, produced by FujiDevison Chemical Ltd.), followed by development with ethylacetate/methanol/water (3:2:1, v/v). The active fraction thus eluted wasconcentrated, and the concentrate was applied to a column packed with125 ml of silica gel (Lichroprep-Si 60, Art 9390; produced by Merck &Co., Inc.), followed by development with ethanol/water (100:3, v/v). Theactive fraction eluted was concentrated to dryness to give 20 mg of UCE1022 as red powder.

Example 2

Paecilomyces sp. SPC-13780 strain (FERM BP-2256) was used as the seedstrain. The strain was inoculated into 300 ml of a seed medium havingthe following composition in a 2-l Erlenmeyer flask, and cultured withshaking (200 rpm) at 30° C. for 48 hours.

Composition of the seed medium (in 1 l): 5 g of peptone, 5 g of dryyeast "Ebios", 10 g of glucose, 200 ml of V8 vegetable juice, 0.5 g ofMg₃ (PO₄)₂ ·8H₂ O (pH 6.0 before sterilization)

The resulting seed culture was transferred into 15 l of a fermentationmedium having the following composition in a 30-l jar fermentor at therate of 5% (by volume), and culturing was carried out at 30° C. withstirring and aeration (rotation: 400 rpm, aeration: 15 l/min.).

Composition of the fermentation medium (in 1 l): 35 g of glycerol, 35 gof glucose, 15 g of dry yeast, 0.5 g of KH₂ PO₄, 0.5 g of MgSO₄ ·7H₂ O,0.5 g of Mg₃ (PO₄)₂ ·8H₂ O (pH 7.0 before sterilization, adjusted withNaOH)

Culturing was carried out for 80 hours without controlling the pH of themedium.

After filtration of the resulting culture, 5 l of acetone was added tothe obtained cells, followed by stirring. The removal of the cells andprecipitates by filtration gave 5 l of a filtrate. To the filtrate wereadded 15 l of ethyl acetate and 0.5 l of ammonia. After stirring, theresulting mixture was passed through a column packed with 2 l of silicagel to adsorb the active substance. The active substance was eluted withacetone/ethyl acetate/ammonia (1:1:0.05, v/v). The active fraction wasconcentrated, and the concentrate was diluted with a 10 mM phosphatebuffer (pH 8.0) and passed through a column packed with a polystyreneadsorption resin, Diaion HP20 (produced by Mitsubishi Kasei Corporation;400 ml) to adsorb the active substance. After impurities were elutedwith a 10 mM phosphate buffer (pH 8.0) and subsequently with 40%methanol containing the buffer of the same concentration, the activesubstance was eluted with 60% methanol. The active fraction wasconcentrated, and the concentrate was diluted with a 10 mM phosphatebuffer (pH 8.0) and subjected to column chromatography using apolystyrene adsorption resin, Diaion HP20SS (produced by MitsubishiKasei Corporation; 200 ml). The 60% methanol-eluted solution wasconcentrated, and the concentrate was dissolved in a small quantity of amixture of methanol and acetone. The active substance was precipitatedfrom the solution by adding hexane. The precipitate was separated byfiltration and dried to give 33 mg of Saintopin E as bluish purplepowder.

Industrial Applicability

According to the present invention, Saintopin derivatives which haveantibacterial and anti-tumor activity can be provided.

We claim:
 1. Saintopin derivatives which are represented by thefollowing formula (I): ##STR4## wherein R¹ is hydrogen and R² is SO₂ OH,or R¹ is acetyl and R² is H.